KR100880509B1 - A Novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same - Google Patents

Abstract

Disclosed herein is an inducible high-expression cassette comprising a dihydrofolate reductase (DHFR) promoter from which GC-rich repeat sequences are partially or entirely removed, the cassette capable of more effectively improving a gene amplification system. Also disclosed are an expression vector comprising the inducible expression cassette and optionally a gene encoding a recombinant protein of interest, an animal cell line transformed with the expression vector, and a method of mass producing and purifying a recombinant protein by culturing the transformant. The present invention enables the shortening of the time required to establish a cell line producing a recombinant protein of interest at high levels using a low concentration of a DHFR inhibitor, thereby allowing more effective production of the recombinant protein.

Description

A novel vector and expression cell line for mass production of recombinant protein and a method for producing recombinant protein using the same

1 is a schematic diagram of a cloning process of expression vectors X1GC / dhfr, X3GC / dhfr and X6GC / dhfr from which GC-rich sequences are sequentially removed.

2 is a schematic diagram of the cloning process of the expression vector X0GC / dhfr from which the GC-rich sequence was completely removed.

Figure 3 is a schematic diagram of a process for cloning the gEPO gene in the expression vector of Figures 1 and 2.

4 to 7 are measured by ELISA of the amount of EPO expression of each cell line transformed with the expression vector of the present invention.

8 shows the result of Western blotting of the expression level of the amplified dihydrofolate gene.

9 is a result of measuring the expression level by indirect ERISA method by recovering the human erythropoietin expression supernatant over a total of nine times by culturing the monoclonal cell line X0GC / GEPO9647 (DXB11) selected in Example 6.

Figure 10 shows the results of SDS-PAGE and Western blotting of the recombinant erythropoietin purified in Example 8.

11 is an isoelectric focusing analysis result of the recombinant erythropoietin purified in Example 4.

12 is an isoelectric focusing result of erythropoietin expressed by culturing X1GC / GEPO9629 (DG44) and X0GC / GEPO9603 (DG44) fully adapted to serum-free medium.

The present invention relates to a vector for mass production of a recombinant protein and an expression cell line thereof, and a method for producing and purifying the recombinant protein using the same. More specifically, a vector, which can greatly improve the amplification efficiency of a gene by artificially weakening a promoter, a transcriptional regulatory region of Dihyrofolate redutase, can be used to prepare a transformed animal cell line. The present invention relates to a method of expressing a protein using an animal cell line and purifying only a high glycated chain form from the expressed protein.

Various vectors and hosts have been used to produce a large amount of recombinant protein, and generally, a method using Escherichia coli is widely used. However, in the case of a protein having glycosylation or a complex structure, its use is limited. To solve these problems, animal cell lines may be used, or yeast, transgenic animals, or transgenic plants may be used.

Although yeast has the advantage of easy mass production, it is known that the degree and pattern of glycosylation is very different from humans, causing many immunogenicity (Hermeling et al, Pharm. Res. 21 (6): 897). -903 (2004)). In the case of transgenic animals, it has not yet been commercialized due to problems related to the management of the animal itself and the possibility of contamination with pathogens.

On the other hand, the method of using animal cell line is the most widely used in the production of recombinant protein because the production cost is high and it takes much time and cost to manufacture the cell line, because the recombinant protein produced is very similar to human form and stable production and management are possible. It is becoming. However, in attempting transformation to produce recombinant protein in animal cell lines, it is difficult to show high productivity due to the small amount of expression. Therefore, in order to solve these problems, a gene amplification method using a cell line is used. The most representative one is a gene amplification method using dihydrofolate reductase or glutamine synthetase, which can greatly increase the yield of recombinant protein and is widely used. have. This gene amplification technology has the advantage of improving productivity, but it takes a lot of time to manufacture by going through several stages of gene amplification to use high concentration of methotrexate (hereinafter, abbreviated as MTX), and long-term passage of cell lines This is known to have problems such as loss of genes and unstable expression.

In order to remedy this drawback, as a dihydrofolate gene promoter, a promoter made by removing a part of the SV40 promoter may be used (Korean Patent No. 0162021), or a mutation may be introduced into a sequencing Cytomegalo virus (CMV) promoter. And a method for controlling affinity for methylated DNA binding protein (Korean Patent No. 0493703) and the like have been reported. Occasionally, the 5'-noncoding region of an immunoglobulin heavy chain binding protein is used as an internal ribosome entry site (IRES) without the use of a single promoter of the dihydrofolate gene, allowing it to be regulated by transcriptional regulators of recombinant proteins. Method (Korean Registered Patent No. 0184778) and the like have been reported.

In the case of Korean Patent Registration No. 0162021, the activity of the promoter was controlled by removing 128-270 sites of the SV40 transcriptional regulator, but the role of the removed sequence is not clear and does not mention the role of the remaining sequence. In particular, since no amplification is no longer performed at concentrations of 20 nM or more under the condition of no control setting, any change in the existing dihydrofolate promoter activity has occurred, and it is optimal to use a low concentration of MTX. Evidence does not provide evidence of the expression of. Korean Patent No. 0493703 attempts to efficiently amplify a gene by changing the binding sequence for the methylated DNA binding protein of the CMV promoter, but the effect of the amplification on the expression is weak compared to other methods. In addition, even when expressed using an internal ribosome entry site (IRS) sequence without using a single promoter as in Korean Patent No. 0148778, the concentration of MTX used for gene amplification is only a few μM, The increase also remained at about 30 times. As described above, the modification of the general promoter cannot be expected due to the amplification of the dihydrofolate gene, and the applicability thereof can be judged only when the gene amplification is performed using various kinds of promoters.

The human erythropoietin exemplified as the recombinant protein of the present invention has a molecular weight of about 18000 Da when the pure molecular weight is about 18000 Da, or about 34000 Da when glycosylated. They are synthesized in the kidneys when anemia, low oxygen partial pressure in the air, or bleeding occurs, thereby promoting red blood cell production and maintaining homeostasis. It is present at a concentration of about 10-20 mIU / mL in normal humans and causes severe anemia when abnormalities occur in the kidneys (Jacobson, et al., Nature, 179: 633-634 (1957)). Therefore, erythropoietin has been used as a therapeutic agent for chronic kidney failure and anemia due to various causes. In the past, they were separated from the blood or urine of patients with diseases such as plasma of animals or aplastic anemia that produces higher concentrations of erythropoietin than normal ones, but they are low in stability and lacking in quantity. In the case of erythropoietin, the concentration of the erythropoietin is low and there is an erythropoietin activity inhibitor, which requires a high-purity purification to remove it (see US Patent No. 4397840, 4303650, 3865810). Since it is difficult to obtain pure high yield of erythropoietin by this method, methods using genetic recombination have been developed. However, erythropoietin has a problem in that glycosylation is essential for in vivo activity, and thus, when the erythropoietin gene is cloned and expressed in Escherichia coli or yeast, glycosylation does not occur properly and erythropoietin bioactivity does not appear. Therefore, production of erythropoietin requires a method using a recombinant animal cell line. Methods using recombinant animal cell lines are indispensable. In the production of erythropoietin using recombinant animal cell lines, gene amplification techniques using the above-mentioned dihydrofolate reductase are generally used (Malik et al. DNA and Cell Bio., 6: 453-459). (1992).

Based on this fact, the inventors of Michel Fromm and Paul Berg (J. Mol. Appl) found that the role of six GC-rich repeats in the promoter region linked to the dihydrofolate structural gene is important for transcriptional activity. Genet. 1983. 2 (1): 127-135), by sequential removal of the GC-rich repeat sequence to produce an animal cell expression vector that gene amplification and expression more efficiently to complete the present invention. .

Accordingly, an object of the present invention is to provide an expression vector capable of amplifying genes more effectively in producing recombinant proteins in animal cells.

Another object of the present invention is to provide an animal cell line transformed with the expression vector.

Another object of the present invention to provide a method for mass production of recombinant protein using the animal cell line.

In order to achieve the above object, the present invention provides an expression vector for an animal cell line comprising a GC-rich repeat deletion promoter of dihydrofolate reductase.

As mentioned above, gene amplification by dihydrofolate reductase has been widely used as a method for inducing high expression of recombinant proteins in animal cell lines, but according to the prior art, dihydrofolate reductase inhibitors. Cell line stabilization and time and cost problems caused by using a high concentration for a long time, the present invention is a dihydrofolate mutated by deleting part or all of the GC-rich repeat sequence of the promoter of such dihydrofolate reductase The expression vector using the reductase gene can express the expression of the dihydrofolate reductase and / or the target recombinant gene included in the vector with higher efficiency even when a lower dihydrofolate reductase inhibitor is used in a shorter time. It was found to be inducible.

Here, the GC-rich repeat sequence refers to a CCGCCC repeat sequence included in a promoter which is a transcription regulator of dihydrofolate reductase, and artificially deleted all or part of the repeat sequence by a method such as deletion or mutation. This results in minimal expression of dihydrofolate reductase. At this time, when the dihydrofolate inhibitor is added while the expression is kept to a minimum, the cells amplify a larger number of dihydrofolate reductase genes for survival, and thus, the dihydrofolate reduction It was observed that the target recombinant gene contained in the expression vector containing the enzyme gene was also amplified and highly expressed at the same time.

Accordingly, in one specific aspect of the present invention, there is provided a high expression induction cassette comprising a nucleotide sequence of the dihydrofolate reductase gene comprising a promoter from which one or more CCGCCC repeats have been removed. Preferably, the high expression induction cassette comprises a promoter of dihydrofolate reductase comprising 6 or less CCGCCC repeats, more preferably a promoter comprising 3 or less CCGCCCC repeats, Particularly preferably it comprises a promoter comprising at most one CCGCCC repeat sequence, more particularly preferably a promoter from which all of the CCGCCC repeat sequence has been removed.

Removal of these CCGCCC repeat sequences may be performed by a method such as substitution or deletion of nucleotide sequences according to gene recombination techniques well known in the art, and in a specific embodiment of the present invention, Some or all of the GC-rich sequences in the promoter were removed by a method of deleting some.

In another aspect of the present invention, an expression vector comprising the high expression induction cassette is provided.

The term 'vector' includes means for expressing a gene of interest in a host cell, including plasmid vectors, cozmid vectors, bacteriophage vectors and adenovirus vectors, retroviral vectors, viral vectors such as adeno-associated viral vectors, and the like. Is a plasmid vector.

Preferably, the expression vector may further include a gene encoding a recombinant protein of interest, and by expressing such expression vector, the recombinant protein of interest may be expressed with high efficiency.

The target recombinant protein generally includes a physiologically active polypeptide, and includes hormones, cytokines, interleukins, interleukin binding proteins, enzymes, antibodies, growth factors, transcription regulators, blood factors, vaccines, structural proteins, and ligand proteins. Or various proteins such as receptors, cell surface antigens, receptor antagonists, and derivatives and analogues thereof, specifically human growth hormone, interferon and interferon receptors, colony stimulating factors, interleukins, erythropoietin, insulin , Angiotensin, bone formation growth factor, B cell factor, T cell factor, nerve growth factor, cell surface antigen, monoclonal antibody and virus derived vaccine antigen and the like. One skilled in the art can readily select recombinant proteins to which amplification techniques by dihydrofolate reductase can be applied at the current state of the art. In a preferred embodiment of the invention, said recombinant protein is human erythropoietin.

The recombinant protein of interest may be expressed by a promoter of the dihydrofolate reductase gene or the expression may be controlled by a separate promoter. Preferably, the recombinant protein of interest is regulated by a separate promoter. Such promoters are well known in the art and can be easily selected and used by those skilled in the art, for example, from the group consisting of cytomegalovirus (CMV) promoter, LTR promoter, EFα promoter, SV40 promoter and TK promoter. It is not limited to.

The expression vector of the present invention is for inducing high expression in animal cell lines, and may further comprise a resistance gene for animal cell lines, which is preferably used as a selection marker for permanent expression in animal cells. The animal cell line resistance genes include neomycin resistance genes, zeocin resistance genes, hygromycin resistance genes, and blocystine resistance genes, which are commonly used in the art, such as animal cell line resistance genes, and the like. no.

In addition, the expression vector of the present invention may further include, but is not limited to, components of a general vector, such as origin of replication and multiple adenylation signals and other transcriptional regulators.

In another aspect of the invention, there is provided a cell line transformed with the expression vector according to the invention.

In a specific embodiment, the present invention prepared a high expression induction cassette having only one CCGCCC repeat sequence and a high expression induction cassette containing no CCGCCC repeat sequence in the promoter of dihydrofolate reductase, respectively, and include these expression cassettes. To provide an E. coli cell line transformed with an expression vector. These cell lines were deposited on October 2, 2006 as Accession Nos. KCTC 10991 BP and KCTC 10992 BP, respectively, to the Gene Bank in the Biotechnology Research Institute, Yuseong-gu, Daejeon, Korea. In order to induce high expression of the desired recombinant protein, these cell lines are separated from the expression vector containing the high expression induction cassette from the cell line and cloned by a gene recombination technique or the like, thereby encoding a gene encoding the target recombinant protein. It can be used for the production of an expression vector further comprising.

In another specific embodiment of the present invention, the expression vector further comprises a gene encoding a recombinant protein of interest, and also provides a cell line transformed by such expression vector.

In a preferred embodiment, these desired recombinant proteins require expression in animal cell lines, and in view of these purposes, preferred animal cell lines usable in the present invention include Chinese hamster ovarian cacer (CHO) cell lines, monkeys. Renal cells 7 (COS7: Monkey kidney cells) cell line, NSO cell line, SP2 / 0 cell line, W138, baby hamster kidney (BHK) cell line, MDCK, myeloma cell line, HuT 78 cells and 293 cells, etc. Are not limited thereto. Those skilled in the art will be able to readily select suitable animal cell lines applicable to the amplification technique using the dihydrofolate reductase of the present invention.

In a specific embodiment of the present invention, a CHO cell line was used as the animal cell line, and more specifically, a Chinese hamster ovary cell line (CHO / dhfr-) deficient in dihydrofolate reductase was used. That is, a CHO cell line deficient in dihydrofolate reductase is transformed into an expression vector according to the present invention comprising a gene encoding human recombinant erythropoietin, so as to have a low methotrexate of 100 nM or less, more preferably 50 nM or less. Sufficient genes are amplified at concentrations to provide animal cell lines with proven productivity. These cell lines, as described in detail in Examples below, were deposited on October 2, 2006 as Accession Nos. KCTC 10993BP, KCTC 10994BP and KCTC 10995BP to the Gene Bank in Biotechnology Research Institute, Yuseong-gu, Daejeon, above.

In another embodiment of the present invention, a gene encoding a dihydrofolate reductase comprising a promoter in which the GC-rich sequence according to the present invention is partially or wholly removed, and a gene encoding a recombinant protein of interest. Transforming the animal cell line with an expression vector for the animal cell; And it provides a method for producing a recombinant protein comprising the step of culturing the transformed animal cell line.

In the present invention, "transformation" into an animal cell line includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ, and is performed by selecting a suitable standard technique according to the animal cell line to be used as known in the art. can do. For mammalian cells without cell walls, calcium phosphate precipitation can be used (Graham et al, 1978, Virology, 52: 456-457). General methods and features of transformation into mammalian host cells are described in US Pat. No. 4,399,216. Specifically, in the present invention, a vector expressing a recombinant protein was transformed into cells using lipofectamine in CHO cells. In addition, the culture of the animal cell line in the present invention can be made according to the appropriate medium and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the animal cell line selected. Depending on the cell growth method, suspension culture and adhesion culture are classified into batch, fed-batch and continuous culture according to the culture method. The medium used for culturing must adequately meet the requirements of the particular cell line.

In animal cell culture, the medium contains various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, mineral nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate This includes. These nitrogen sources may be used alone or in combination. In addition, amino acids, vitamins and suitable precursors may be included.

In addition, a dihydrofolate reductase inhibitor such as methoxtrexate may be added to the medium. As described above, the protein recombination method according to a preferred embodiment of the present invention transforms the expression vector according to the present invention to an animal cell line in which the dihydrofolate reductase is deleted, and dihydropol to amplify the recombinant gene. This is because a system for adding a rate reductase inhibitor to allow a dihydrofolate reductase in a vector to be amplified and selected can be efficiently implemented in a short time.

According to a preferred embodiment of the present invention, the dihydrofolate inhibitor is preferably used as short as possible in consideration of the stability and economic efficiency of the cell line. In other words, the use of dihydrofolate reductase inhibitors at low concentrations enables stability in mass production and shorter development cell line development periods. Specifically, the present invention is a method for producing a recombinant protein using methotrexate at a concentration of 100 nM or less, preferably 50 nM or less by transforming the recombinant protein expression vector into a Chinese hamster ovary cell line deficient in dihydrofolate reductase. To provide.

In addition, as an additional aspect of the present invention, when producing erythropoietin from the cell line described above, a large amount of sialic acid in the produced erythropoietin to purify the high sialic acid-containing erythropoietin excellent in physical properties It may further comprise a step.

In an embodiment of the present invention, the gene is amplified by adding a dihydrofolate reductase inhibitor after artificially deleting the GC-rich sequence in the promoter of the dihydrofolate reductase to minimize expression. To obtain clones originating from one of the cell lines subjected to gene amplification, limiting dilution was performed to obtain monoclonal cell lines, which were mass cultured to produce recombinant human erythropoietin in serum-free medium. At this time, the erythropoietin eluted at low salt concentration is a fraction having a high sialic acid content, and the erythropoietin eluted at a high salt concentration is a fraction having a low sialic acid content. Was eluted to purify erythropoietin containing gosialic acid, and activity was confirmed.

The present invention will be described in more detail based on the following examples. This embodiment is only for illustrating the invention in detail, the present invention is not limited to the following examples.

Example  One.

Example  One. GC - rich  Preparation of Expression Vectors with Repeated Sequences Removed

Primer dhfr 01 (5 ') with a SmaI restriction enzyme recognition site from the pSV2-dhfr (ATCC # 37146) plasmid to create a dihydrofolate reductase gene cassette structure expressed by a promoter deleted with a GC-rich repeat sequence -GCG CCC GGG ATG GTT CGA CCA TTG AAC TGC-3 ') and primer dhfr-02 (5'-CAC TTA GAA CCT GTT AGT CTT TCT TCT CGT AGA C-3') with BstBI restriction enzyme recognition site PCR was performed to amplify the dihydrofolate reductase gene, and the obtained amplification product of about 200 bp was electrophoresed on a 1% agarose gel and recovered using QIAGEN's gel extraction kit (cat # 28706). The recovered gene fragment was directly inserted into the pDRIVE vector of Qiagen, cloned, and subjected to sequencing to confirm that there was no error. Dihydrofolate reductase gene was digested with restriction enzyme SmaI / BstBI from the nucleotide sequence pDRIVE-dhfr plasmid, and electrophoresed on 1.5% agarose gel. # 28706). The dihydrofolate reductase gene fragment obtained by this method was replaced by cutting the neomycin gene sequence of Invitrogen's pcDNA 3.1 vector with the same restriction enzyme. Using the cloned pcDNA3.1-dhfr plasmid as a template, the primer x 1GC (5'-TCA GGA TCC ATT) having a BamHI restriction enzyme recognition site and complementary to different GC-rich repeats of the dihydrofolate reductase promoter CTC CGC CCC ATG GCT GAC TAA-3 '), x 3GC (5'-CAT GGA TCC TAA CTC CGC CCA GTT CCG CCC ATT CT-3'), x 6GC (5'-CAT GGA TCC CAT AGT CCC GCC CCT AAC TCC GCC C-3 ') and a primer BISVpAR (5'-TCA GGA TCC CAG ACA TGA TAA GAT), which also has a BamHI restriction enzyme recognition site and is complementary to the polyadenylation signal sequence structurally linked with dihydrofolate reductase PCR using ACA TTG ATG-3 ') yielded three different size gene cassettes with partial loss of GC-rich repeat sequences. The gene cassette was inserted and cloned into the invitrogen pcDNA3.1 vector digested with BglII restriction enzyme, and clones having the same orientation as the CMV promoter of the vector were selected using restriction enzyme mapping. Through this, X1GC / dhfr, X3GC / dhfr and X6GC / dhfr plasmids were prepared. Each plasmid was used to clone the erythropoietin gene from human genomic DNA. In addition, cloning was performed as follows to prepare a control expression vector from which the GC-rich repeat sequence was completely removed.

Primers X0GC (5'-CGA TGG ATC CGA CAT GAT AAG ATA CAT TGA T-3 ') and X0GCRR (5 '-CGT TGG ATC CAC AGC TCA GGG CTG CGA TTT C-3'). PCR was performed using the pSV2-dhfr plasmid as a template to obtain an amplification product of sequence 1.5 kb from the SV40 virus polyadenylate sequence to the 5 'non-toxic site of the dihydrofolate reductase gene. The gene fragment was electrophoresed on a 1% agarose gel and then recovered using a gel extraction kit (QIAGEN, cat # 28706) and inserted at the position where the invitrogen pcDNA3.1 was cut with restriction enzyme BglII. Restriction maps were used to select clones in which the dihydrofolate reductase gene was located in the opposite direction to the CMV promoter. In order to remove several restriction enzyme recognition sites, the polycloning site of the vector was digested with restriction enzymes NdeI and DraIII, and the pRcCMV vector of invitrogen was replaced with a fragment cut in the same manner. X0GC / dhfr vector was produced through this.

The above cloning schematic is summarized in FIG. 1 and FIG.

Example  2. gEPO  Gene Cloning

The pCI-neo / gEPO plasmid was digested with XhoI / EcoRI restriction enzyme, the end of adhesion was blunted with DNA polymerase I Klenow fragment, and then electrophoresed on a 0.7% agarose gel to correspond to the human erythropoietin genome. 2.2Kb DNA fragments were recovered using a gel extration kit (QIAGEN, cat # 28706). The Invitrogen pRcCMV vector was digested with EcoRV and recovered with QIAGEN's PCR purification kit (cat # 28106), and then cloned by attaching the erythropoietin genomic gene to the EcoRV cleavage site. Restriction maps were used to select clones in which the erythropoietin genomic gene of the vector has the same orientation as the CMV promoter. Example 1 X0GC / dhfr, x1GC / dhfr, x3GC / dhfr, x6GC / dhfr vectors were digested with BamHI / XhoI restriction enzymes and subjected to electrophoresis on 1% agarose gels followed by gel extration kit (QIAGEN, cat # 28706). ) Was recovered using. In the same manner, the erythropoietin genomic fragment cloned into the pRcCMV vector was recovered and inserted into the BamHI / XhoI position of the multicloning sites of the cut x0GC / dhfr, x1GC / dhfr, x3GC / dhfr and x6GC / dhfr vectors. It was. The nucleotide sequences of the clones obtained were confirmed to be free of errors and named as X0GC / GEPO, X1GC / GEPO, X3GC / GEPO, and X6GC / GEPO, respectively. The above cloning process is summarized in FIG. 3, and each final sequence is shown in the sequence list.

Example  3 Dihydrofolate  Transformation of Chinese Hamster Ovary Cell Line

Chinese hamster ovary cell lines lacking the dihydrofolate gene, CHO / DXB11 strain and CHO / DG44 strain, were treated with 10% fetal bovine serum (Welgene, Cat # S101-01) and 1% penicillin-streptomycin (Gibco, Inoculated in DMEM / F12 medium (Welgene, Cat # LM002-04) containing Cat # 15140-122) was passaged at 37 ℃, 5% CO ₂ thermostat. 1 × 10 ⁶ cells were placed in a 6 cm diameter cell culture dish to transform each dehydrofolate gene deficient Chinese hamster ovary cell line with the prepared X0GC / GEPO, X1GC / GEPO, X3GC / GEPO and X6GC / GEPO plasmids. Inoculated and incubated for 24 hours at 37 ℃, 5% CO ₂ incubator, washed twice with Gibco's Opti-MEM medium (Gibco., Cat # 31985-070). After mixing 1 mL of Lipofectamine ^TM Reagent (Invitrogen, Cat # 18324-020) in 3 1 mL Opti-MEM containing 10 μg of each plasmid, the reaction was allowed to stand at room temperature for 20 minutes, and then the prepared dihydrofolate gene. Evenly distributed in the defective Chinese hamster ovary cells, incubated for 18 hours at 37 ℃, 5% CO ₂ incubator, then exchanged with DMEM / F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin Incubate for 48 hours. 10% dialyzed fetal bovine serum Welgene (Cat #XXXX), 1% penicillin-streptomycin, and 800 μg / mL geneticin (Mediatech, Cat # 61-234 RG) for selection of transformed cell lines. Cell lines obtained by treatment with 0.5% Trypsin-EDTA (Gibco., Cat # 15400-054) and centrifugation in α-MEM (Welgene, Cat # LM008-02) screening medium were transferred to T 25 cell culture vessel. After inoculation, the cells were cultured at 37 ° C. in a 5% CO ₂ incubator, and cultured so that 90% or more of the cell lines transformed and selected with geneticin were cultured. Cell lines were selected at the same concentrations of geneticin and culture conditions, and it was observed that the screening effect appeared rapidly in cell lines with more GC repeats removed. No significant differences were found between strains of the dihydrofolate gene-deficient hamster ovary cell line.

Example  4. Selection of Recombinant Cell Lines and Amplification of Erythropoietin Gene

Methotrexate (MTX, Sigma, Cat # M-8407) was used to increase the expression level of erythropoietin of each cell line (CHO / DXB11 strain or CHO / DG44 strain) transformed and selected as geneticin. 2 × 10 ⁴ cells / well were inoculated into a 24-well culture vessel in a selection medium (same as the above selection medium) added at a concentration of 20 nM, and then cultured in a 37 ° C., 5% CO ₂ incubator for 2 weeks. In order to select a cell line with high expression amount of erythropoietin, the cell line incubated so that the bottom of the well is 100% covered in a 24-well culture vessel was washed twice with PBS (Welgene, Cat # LB 001-02), and then erythropoie CHO-A-SFM (Gibco. Cat # 05-5072EF), an ethine production medium, was added to 200 μL / well, and the culture supernatant incubated for 24 hours was recovered to recover the indirect Eliza (EPO ELISA kit, R & D, Cat # DEP00). Expression level was measured by the method. After raising methotrexate concentration to 30 nM, the cells were cultured again for two weeks in the same selection medium, and the amount of erythropoietin expression of each cell line was measured by ELISA (Ezyme linked immunosorbent assay) kit (R & D systems cat # DEP00). By comparison. As shown in FIGS. 4-7, X0GC / GEPO and X1GC / GEPO showed the highest expression levels at the same concentration of MTX as shown in FIGS. 4 to 7, followed by X3GC / GEPO and X6GC. Followed by / GEPO. This fact indicates that the more amplification of the GC sequence of the dihydrofolate gene, the more amplification of the gene occurs in the presence of the same concentration of methotrexate. In addition, efficient gene amplification also occurred in X0GC / GEPO where the GC repeat sequence was completely removed. Therefore, it was confirmed that the amplification effect is maintained at the maximum when the GC repeat sequence is kept at the minimum. In addition, in order to confirm the concentration of methotrexate in which gene expression occurs to the maximum, we tried to increase the concentration of methotrexate in the selection medium from 40 nM to 60 nM, but there was no significant increase in expression level or change in acid isomer content. Confirmed. Based on the results, limit dilution was performed on two cell line strains transformed with X0GC / GEPO and X1GC / GEPO as in Example 6.

Example  5. Dihydrofolate  Confirmation of gene amplification

Each cell obtained in Example 4 was washed once with D-PBS solution, treated with trypsin-EDTA, recovered from the culture flask, and centrifuged at 1,000 rpm for 3 minutes to obtain cells. This was washed once again with D-PBS and counted with a hematocytometer (hemacytometer, incyto) to count 3 x 10 ⁶ cells, followed by centrifugation to obtain cell precipitation. The obtained cells were each suspended in 1.5 mL of cell lysis buffer (PBS, 5 mM EDTA, 1% NP-40), lysed at 4 ° C. for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes to obtain a supernatant.

12.5% SDS polyacrylamide gel was prepared, and each cell lysate and negative control CHO / dhfr-cell lysate were taken in 20 uL each, mixed with 10 uL of sample buffer and pre-treated at 100 ° C. for 5 minutes to be loaded onto the gel. . As a positive control, 20 ng to 100 ng of dihydrofolate reductase (Sigma, cat # D6566) was mixed with sample buffer and loaded onto the gel. After the loaded gel was electrophoresed at a current of 30 mA, the gel was separated and superimposed with a PVDF filter and 3MM paper in a semi-pore western blotting unit (SEMI-PHOR, Hoefer Co., Ltd.). Moved to PVDF room. Then, the protein-transferred PVDF space was separated, and anti-dihydrofolate reductase mouse antibody (BD Biosciences, cat # 610697) was 1: 500 in 10 mL of TBS-T solution containing 0.5% skim milk. The reaction was stirred for 1 hour in the added solution. After washing 10 times with TBS-T solution 5 times, anti-mouse antibody labeled with horseradish peroxidase in 10 ml of TBS-T solution containing 0.5% skim milk (Amersham Biosciences, cat # RPN2108) Included in the ratio of 1: 3000, and reacted with stirring for 1 hour. After washing 5 times with TBS-T solution for 5 minutes each, one and two agents of ECL Western Blotting Analysis System (ECL Western Blotting Analysis System, Amersham Biosciences, cat # RPN2108) were mixed in a 1: 1 ratio to allow for PVDF. Sprinkle on top and allowed to react for 1 minute. Immediately after the reaction, the photosensitive film was developed with an X-ray film and developed. Western blotting results are shown in FIG. 9. As can be seen from the results of FIG. 9, it was found that the expression level of the dhfr gene was increased by transformation. In the same cell number, dhfr expression was higher in X0GC or X1GC / GEPO.

Example  6. Monoclonal  Isolation and Selection of Cell Lines

The X0GC / GEPO and X1GC / GEPO cell lines of the wells, which had the highest expression amount in Example 4, were transferred to 6-well culture vessels and incubated, and methotrexate was added to the 96-well culture vessels at 0.5 cells per well to separate monoclonal cells. Each cell line was inoculated at the limiting dilution with the added selection medium. Incubate at 37 ° C. in a 5% CO ₂ incubator for about 2 to 3 weeks, select only the wells from which single colonies have been produced, transfer them to 24-well culture vessels, and incubate the erythropoietin expression in an indirect IRIS method as in Example 4. Measured.

Of these, monoclonal X0GC / GEPO9647 (DXB11) clones and X1GC / GEPO9629 (DG44) and X0GC / GEPO9603 clones with high expression and high ratios of acid isomers in isoelectrically focused isomers were isolated and selected as the final monoclonal cell lines. The expression levels and isoelectric focusing isoforms of the monoclones were analyzed.

In the case of X0GC / GEPO9647 (DXB11), the expression level per cell line was about 80 μg / 10E6 cells / day. As described above, in the case of X0GC / GEPO or X1GC / GEPO used in this example, a method capable of obtaining a high expression cell line by only two stages of amplification of the gene enables production of a monoclonal cell line for protein production with only a low passage. It is showing that it is possible.

Example  7 Monoclonal  Mass culture of cell lines

In order to mass-produce human erythropoietin, amplification of X0GC / GEPO9647 (DXB11) among the monoclonal cell lines selected in Example 6 in a T175 culture vessel in stages was carried out in steps of amplification up to 120 T175 flasks in total. Approximately 2.5 X 10 ⁸ cell lines were inoculated per factory (Nunc, Cat # 170009), and after inoculating a total of eight cell factories, the cells were incubated at 37 ° C. and 5% CO ₂ incubator for about 48 hours. After washing twice with 1 liter per Cell Factory in phosphate buffer, serum-free CHO-A-SFM (Gibco Fomula #), a medium for the production of erythropoietin with 0.3 mM sodium butyrate (Sigma, Cat. B-5887), was added. 05-5072EF) in 1 liter each, 33 ℃, 5% CO ₂ Human erythropoietin-expressing supernatants were harvested for 9 times every 2 days while incubating in a thermostat. The recovered supernatant was removed using a centrifuge and a pore size 0.2 μm filter paper to remove solid suspensions such as cell residues. A portion of the recovered supernatant was taken, and the expression level was measured by indirect ELISA method, and isoelectric focusing was performed to analyze the contents of acidic isoelectric focusing variants. As shown in the results of FIG. 10, it can be seen that the expression level of 40-50 mg / L is maintained even in a large amount of culture, and the contents of isomers 6 to 8 which can be used as a measure of high sialic acid content are separately purified. Although it did not go through the process was found to be high.

Example  8 Gosialic acid  Purification of Containing Human Erythropoietin

In order to remove the suspended solids in the cell culture produced in Example 7, using a Beckman XL-90 centrifuge, centrifugation at 7,000 rpm in a JLA-8.1000 rotor, the supernatant was recovered and filtered through a 0.2 um filter paper. The ultrafiltration membrane (ultrafiltration membrane) was used to concentrate to 1/10 volume. To the concentrate was mixed the same amount of 20 mM Sodium Phosphate pH 7.4 buffer. Amersham Blue FF column was equilibrated with 20 mM Sodium Phosphate pH 7.4 buffer, the above erythropoietin-containing solution was applied, washed with 5 mM volume of column with 20 mM Sodium Phosphate pH 7.4 buffer, 2 M NaCl, 20 Erithropoietin was eluted by applying a concentration gradient of 2 times the column volume using mM Sodium Phosphate pH 7.4 buffer as the B buffer.

The erythropoietin fraction was the main fraction, and the desalting and pH of the solution were changed to 20 mM Sodium Phosphate pH 5.4 buffer of the erythropoietin-containing solution using Sephardex G25 column. This erythropoietin-containing solution was applied to Amersham's SP HP column equilibrated with 20 mM Sodium Phosphate pH 5.4 and wiped by 5 times column volume with 20 mM Sodium Phosphate pH 5.4 buffer, followed by 1 M NaCl, 20 mM Sodium Phosphate pH. 7.4 erythropoietin was eluted by applying a 12-fold column volume concentration gradient with buffer as B buffer. At this time, the erythropoietin eluted at a low salt concentration is a fraction with high sialic acid content, and the erythropoietin eluted at a high salt concentration is a fraction with a low sialic acid content. Only the remaining part was taken except for the part corresponding to%. The erythropoietin fraction purified on the SP HP column was then removed using a Sephardex G25 column equilibrated with 10 mM Tris, pH 7.5 buffer to remove salts and change the buffer. This was applied to Amersham's Source 15 Q column equilibrated with 10 mM Tris pH 7.5 buffer and erythropoietin was eluted by applying a six-fold column volume concentration gradient with 0.25 M NaCl, 10 mM Tris, pH 7.5 buffer to B. . Fractions of erythropoietin eluted at least 15, each fraction was analyzed by the charge isomer distribution using SDS-PAGE, isoelectric focusing and capillary zone electrophoresis (CZE), free from impurities The highest salt concentration fraction was collected from the fraction containing the most erythropoietin with 10 acid contents. As a result of the above purification process, the productivity of recombinant human erythropoietin with a sialic acid content of 10 mol or more reached about 20 μg / mL.

Example  9 Of human erythropoietin  Property check

① SDS - PAGE  And Weston Blotting  analysis

The recombinant erythropoietin purified in Example 8 described above was subjected to electrophoresis on two 12% SDS-PAGE, and one gel was observed by staining and decolorizing in Comash Brilliant Blue staining solution. The other gel was adsorbed onto the PVDF membrane (Roche, cat #) using a semi-dry electric transition machine and treated with anti-human EPO antibody (R & D systems cat # AB-286-NA) at 1: 5,000 and washed. And anti-mouse rabbit antibody conjugated with alkaline phosphatase (Amersham cat # NA934V). After washing sufficiently with phosphate buffer containing 0.5% Tween 20, it was treated with Amersham's coloring reagent (cat # RPN2108), and then subjected to photosensitive film in the dark, and developed using an automatic developing machine. As a result, it was confirmed that the band of erythropoietin was SDS-PAGE, and the size difference was shown depending on the degree of glycosylation (see FIG. 10).

② Isoelectric Focus  analysis

The cell culture concentrate and the recombinant erythropoietin purified in Example 4 above were mixed with the sample buffer (Invitrogen cat # LC5371) in 1: 1 and then BRP (BRP cat # E1515000) in an isoelectric focusing gel (Invitrogen cat # EC6655B). They were loaded with standard and isoelectric focusing materials and electrophoresed in turn at low and high voltages. After electrophoresis, the isoelectric focusing gel was immobilized by shaking for 30 minutes in a fixed solution containing 12% TCA and 3% sulfosalisylic acid, and stained with Coomas Brilliant Blue R250 (Amresco cat # 6104-59-2). After staining at erythropoietin corresponding to each isoelectric point was observed. (See Figure 11)

③ TF With -1 cell line Activity and  Activity analysis

TF-1 (ATCC cat #) grown on RPMI 1640 (Welgene cat # LM 011-03) containing 10% fetal bovine serum, 10 uM β-mercaptoethanol, 20 ug / mL transferrin, and 12 ng / mL GM-CSF CRL-2003) is recovered by centrifugation at 1,000 rpm for 5 minutes. The recovered TF-1 cell line was washed twice with phosphate buffer and assayed with 2% fetal bovine serum and 100 ug / mL transferrin, 2 mg / mL protease-free bovine serum, 1% penicillin / streptomycin. One wash was performed with the medium. 50 uL of assay medium was added to a 96 well plate, and 25 uL of sample and standard product were added to the starting well to make a final concentration of 1 ug / mL, and then serially diluted three times. The TF-1 cell line subjected to washing with assay medium to each well, 2 X 10 ^4/50 was further causes the uL / well. After reacting for about 72 hours in a 37 ° C. 5% CO ₂ incubator, 20 uL of each color well of Promega's cell titer one solution kit (cat # G4102) was added. After 4 hours of color development at 37 ° C. in a 5% CO ₂ incubator, the mixture was mixed well and the absorbance at 490 nm was measured in an ELISA reader manufactured by Molecular Dynamics. Through this test, it was confirmed that the recombinant erythropoietin activity derived from X0GC / GEPO was not different from the standard product.

Example  10 Monoclonal  Cell line Serum-free medium adaptation

X1GC / GEPO9629 (DG44) and X0GC / GEPO9603 (DG44) strains with 10% dialyzed fetal bovine serum, 1% penicillin-streptomycin, 800 ug / mL of geneticin (Mediatech, Cat # 61-234 RG A-MEM (Welgene, Cat # LM008-02) selection medium containing) was used to allow more than 90% culture on the bottom in two T175 culture vessels. After treatment with 0.5% trypsin (Gibco., Cat # 15400-054), the cell precipitated mass obtained by centrifugation was suspended with 10 ml of PBS (Welgene, Cat # LM001-01), followed by centrifugation again, and then the cells at the bottom. The mass was suspended in EXH-CELL CD CHO (cat # 14360) culture medium from JRH Company with 8 mM glutamine. Cell culture medium suspended in serum-free medium was placed in a T175 culture vessel and CO ₂ Stationary culture was performed in the incubator. After about 3 days of incubation, the floating cells that did not adhere to the bottom were centrifuged, transferred to a new T25 culture vessel, and then stationed again for 9 days. Nine days later, the cells were removed from the culture vessel again, and the number of cells, growth curve, and survival rate were measured. The same operation was repeated until cell division was performed within 24 hours and the survival rate was 80% or more. To confirm the expression pattern of X0GC / GEPO9603 (DG44) cell line and X1GC / GEPO9629 (DG44) fully adapted with serum-free medium, 200 mL culture medium at a concentration of 1.0 X 10E5 cells / mL in 500 mL spinner culture vessel (Bellco). Stirring culture was performed at 50 rpm. After 7 days of culture, 100 mL of the culture medium was changed to a new culture medium, and the low temperature expression was performed by lowering the culture temperature to 33 ° C. Culture supernatants were taken at 24 hour intervals to perform isoelectric focusing to determine the acid isomer content of erythropoietin expressed. As shown in FIG. 12, X0GC / GEPO9603 (DG44) and X1GC / GEPO9629 (DG44) fully adapted to serum-free medium showed an isoelectric focusing profile comparable to that of erythropoietin produced in the existing cell factory. Could confirm.

In the case of using the GC-rich sequence-deleted vector and cell line of the dihydrofolate reductase according to the present invention, short-term use of a low concentration of the dihydrofolate reductase inhibitor to select a cell line producing human erythropoietin It is possible to shorten the development period of the cell line producing erythropoietin at high concentration, and to use the cell line to mass produce the desired recombinant protein with high efficiency, gene amplification technology which is one of the methods used in the existing animal cell expression vector. There is an effect to improve more efficiently.

<110> Hanmi Pharm. Co., Ltd. <120> A Novel vector and expression cell line for mass production of          recombinant protein and a process of producing recombinant          protein using same <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 9227 <212> DNA <213> Artificial Sequence <220> <223> X0GC / GEPO vector <400> 1 gacggatcgg gagatccgac atgataagat acattgatga gtttggacaa accacaacta 60 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 120 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 180 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg 240 ctgattatga tctctagtca aggcactata catcaaatat tccttattaa cccctttaca 300 aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag acactctatg 360 cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc tacttataaa 420 ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt cctttgtggt 480 gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca aaaaacttag 540 caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg gaggagtaga 600 atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg agcaaaacag 660 gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat aggttggaat 720 ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta aaaattttat 780 atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca caccacagaa 840 gtaaggttcc ttcacaaaga tccaaagcca gcaaaagtcc catggtctta taaaaatgca 900 tagctttagg aggggagcag agaacttgaa agcatcttcc tgttagtctt tcttctcgta 960 gacttcaaac ttatacttga tgcctttttc ctcctggacc tcagagagga cgcctgggta 1020 ttctgggaga agtttatatt tccccaaatc aatttctggg aaaaacgtgt cactttcaaa 1080 ttcctgcatg atccttgtca caaagagtct gaggtggcct ggttgattca tggcttcctg 1140 gtaaacagaa ctgcctccga ctatccaaac catgtctact ttacttgcca attccggttg 1200 ttcaataagt cttaaggcat catccaaact tttggcaaga aaatgagctc ctcgtggtgg 1260 ttctttgagt tctctactga gaactatatt aattctgtcc tttaaaggtc gattcttctc 1320 aggaatggag aaccaggttt tcctacccat aatcaccaga ttctgtttac cttccactga 1380 agaggttgtg gtcattcttt ggaagtactt gaactcgttc ctgagcggag gccagggtag 1440 gtctccgttc ttgccaatcc ccatattttg ggacacggcg acgatgcagt tcaatggtcg 1500 aaccatgatg gcagcgggga taaaatccta ccagccttca cgctaggatt gccgtcaagt 1560 ttggcgcgaa atcgcagccc tgagctgtgg atctcccgat cccctatggt gcactctcag 1620 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 1680 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 1740 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 1800 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 1860 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 1920 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 1980 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 2040 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 2100 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 2160 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 2220 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 2280 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 2340 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg 2400 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga 2460 gacccaagct tggtaccgag ctcggatcca ctagtaacgg ccgccagtgt gctggaattc 2520 tgcagattcg aggtcgacgg tatcgataag cttccaagcg cggagatggg ggtgcacggt 2580 gagtactcgc gggctgggcg ctcccgcccg cccgggtccc tgtttgagcg gggatttagc 2640 gccccggcta ttggccagga ggtggctggg ttcaaggacc ggcgacttgt caaggacccc 2700 ggaaggggga ggggggtggg gcagcctcca cgtgccagcg gggacttggg ggagtccttg 2760 gggatggcaa aaacctgacc tgtgaagggg acacagtttg ggggttgagg ggaagaaggt 2820 ttgggggttc tgctgtgcca gtggagagga agctgataag ctgataacct gggcgctgga 2880 gccaccactt atctgccaga ggggaagcct ctgtcacacc aggattgaag tttggccgga 2940 gaagtggatg ctggtagctg ggggtggggt gtgcacacgg cagcaggatt gaatgaaggc 3000 cagggaggca gcacctgagt gcttgcatgg ttggggacag gaaggacgag ctggggcaga 3060 gacgtgggga tgaaggaagc tgtccttcca cagccaccct tctccctccc cgcctgactc 3120 tcagcctggc tatctgttct agaatgtcct gcctggctgt ggcttctcct gtccctgctg 3180 tcgctccctc tgggcctccc agtcctgggc gccccaccac gcctcatctg tgacagccga 3240 gtcctggaga ggtacctctt ggaggccaag gaggccgaga atatcacggt gagacccctt 3300 ccccagcaca ttccacagaa ctcacgctca gggcttcagg gaactcctcc cagatccagg 3360 aacctggcac ttggtttagg gtggagttgg gaagctagac actgcccccc tacataagaa 3420 taagtctggt ggccccaaac catacctgga aactaggcaa ggagcaaagc cagcagatcc 3480 tacggcctgt gggccagggc cagagccttc agggaccctt gactccccgg gctgtgtgca 3540 tttcagacgg gctgtgctga acactgcagc ttgaatgaga atatcactgt cccagacacc 3600 aaagttaatt tctatgcctg gaagaggatg gaggtgagtt cctttttttt tttttttcct 3660 ttcttttgga gaatctcatt tgcgagcctg attttggatg aaagggagaa tgatcgaggg 3720 aaaggtaaaa tggagcagca gagatgaggc tgcctgggcg cagaggctca cgtctataat 3780 cccaggctga gatggccgag atgggagaat tgcttgagcc ctggagtttc agaccaacct 3840 aggcagcata gtgagatccc ccatctctac aaacatttaa aaaaattagt caggtgaagt 3900 ggtgcatggt ggtagtccca gatatttgga aggctgaggc gggaggatcg cttgagccca 3960 ggaatttgag gctgcagtga gctgtgatca caccactgca ctccagcctc agtgacagag 4020 tgaggccctg tctcaaaaaa gaaaagaaaa aagaaaaata atgagggctg tatggaatac 4080 attcattatt cattcactca ctcactcact cattcattca ttcattcatt caacaagtct 4140 tattgcatac cttctgtttg ctcagcttgg tgcttggggc tgctgagggg caggagggag 4200 agggtgacat gggtcagctg actcccagag tccactccct gtaggtcggg cagcaggccg 4260 tagaagtctg gcagggcctg gccctgctgt cggaagctgt cctgcggggc caggccctgt 4320 tggtcaactc ttcccagccg tgggagcccc tgcagctgca tgtggataaa gccgtcagtg 4380 gccttcgcag cctcaccact ctgcttcggg ctctgggagc ccaggtgagt aggagcggac 4440 acttctgctt gccctttctg taagaagggg agaagggtct tgctaaggag tacaggaact 4500 gtccgtattc cttccctttc tgtggcactg cagcgacctc ctgttttctc cttggcagaa 4560 ggaagccatc tcccctccag atgcggcctc agctgctcca ctccgaacaa tcactgctga 4620 cactttccgc aaactcttcc gagtctactc caatttcctc cggggaaagc tgaagctgta 4680 cacaggggag gcctgcagga caggggacag atgaccagct tgatatcgaa ttatccatca 4740 cactggcggc cgctcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc 4800 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 4860 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 4920 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 4980 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 5040 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc 5100 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 5160 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 5220 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 5280 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 5340 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 5400 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 5460 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 5520 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc 5580 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 5640 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg 5700 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 5760 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc 5820 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct 5880 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa 5940 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 6000 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 6060 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag 6120 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 6180 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 6240 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 6300 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 6360 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 6420 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat 6480 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 6540 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 6600 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 6660 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 6720 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac 6780 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 6840 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca 6900 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 6960 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 7020 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 7080 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 7140 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 7200 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 7260 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 7320 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 7380 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 7440 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 7500 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 7560 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 7620 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 7680 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 7740 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 7800 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 7860 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 7920 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 7980 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 8040 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 8100 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 8160 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 8220 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 8280 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 8340 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 8400 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 8460 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 8520 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 8580 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 8640 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 8700 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 8760 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 8820 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 8880 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 8940 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 9000 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 9060 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 9120 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 9180 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc 9227 <210> 2 <211> 8635 <212> DNA <213> Artificial Sequence <220> <223> X1GC / GEPO vector <400> 2 gacggatcgg gagatccatt ctccgcccca tggctgacta atttttttta tttatgcaga 60 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 120 cctaggcttt tgcaaaaagc tcccgggatg gttcgaccat tgaactgcat cgtcgccgtg 180 tcccaaaata tggggattgg caagaacgga aacctaccct ggcctccgct caggaacgag 240 ttcaagtact tccaaagaat gaccacaacc tcttcagtgg aaggtaaaca gaatctggtg 300 attatgggta ggaaaacctg gttctccatt cctgagaaga atcgaccttt aaaggacaga 360 attaatatag ttctcagtag agaactcaaa gaaccaccac gaggagctca ttttcttgcc 420 aaaagtttgg atgatgcctt aagacttatt gaacaaccgg aattggcaag taaagtagac 480 atggtttgga tagtcggagg cagttctgtt taccaggaag ccatgaatca accaggccac 540 ctcagactct ttgtgacaag gatcatgcag gaatttgaaa gtgacacgtt tttcccagaa 600 attgatttgg ggaaatataa acttctccca gaatacccag gcgtcctctc tgaggtccag 660 gaggaaaaag gcatcaagta taagtttgaa gtctacgaga agaaagacta acaggttcga 720 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 780 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 840 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 900 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 960 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgggatc tcccgatccc 1020 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc 1080 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg 1140 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt 1200 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa 1260 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 1320 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 1380 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 1440 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 1500 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 1560 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 1620 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 1680 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 1740 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 1800 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat 1860 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt ggtaccgagc 1920 tcggatccac tagtaacggc cgccagtgtg ctggaattct gcagattcga ggtcgacggt 1980 atcgataagc ttccaagcgc ggagatgggg gtgcacggtg agtactcgcg ggctgggcgc 2040 tcccgcccgc ccgggtccct gtttgagcgg ggatttagcg ccccggctat tggccaggag 2100 gtggctgggt tcaaggaccg gcgacttgtc aaggaccccg gaagggggag gggggtgggg 2160 cagcctccac gtgccagcgg ggacttgggg gagtccttgg ggatggcaaa aacctgacct 2220 gtgaagggga cacagtttgg gggttgaggg gaagaaggtt tgggggttct gctgtgccag 2280 tggagaggaa gctgataagc tgataacctg ggcgctggag ccaccactta tctgccagag 2340 gggaagcctc tgtcacacca ggattgaagt ttggccggag aagtggatgc tggtagctgg 2400 gggtggggtg tgcacacggc agcaggattg aatgaaggcc agggaggcag cacctgagtg 2460 cttgcatggt tggggacagg aaggacgagc tggggcagag acgtggggat gaaggaagct 2520 gtccttccac agccaccctt ctccctcccc gcctgactct cagcctggct atctgttcta 2580 gaatgtcctg cctggctgtg gcttctcctg tccctgctgt cgctccctct gggcctccca 2640 gtcctgggcg ccccaccacg cctcatctgt gacagccgag tcctggagag gtacctcttg 2700 gaggccaagg aggccgagaa tatcacggtg agaccccttc cccagcacat tccacagaac 2760 tcacgctcag ggcttcaggg aactcctccc agatccagga acctggcact tggtttaggg 2820 tggagttggg aagctagaca ctgcccccct acataagaat aagtctggtg gccccaaacc 2880 atacctggaa actaggcaag gagcaaagcc agcagatcct acggcctgtg ggccagggcc 2940 agagccttca gggacccttg actccccggg ctgtgtgcat ttcagacggg ctgtgctgaa 3000 cactgcagct tgaatgagaa tatcactgtc ccagacacca aagttaattt ctatgcctgg 3060 aagaggatgg aggtgagttc cttttttttt ttttttcctt tcttttggag aatctcattt 3120 gcgagcctga ttttggatga aagggagaat gatcgaggga aaggtaaaat ggagcagcag 3180 agatgaggct gcctgggcgc agaggctcac gtctataatc ccaggctgag atggccgaga 3240 tgggagaatt gcttgagccc tggagtttca gaccaaccta ggcagcatag tgagatcccc 3300 catctctaca aacatttaaa aaaattagtc aggtgaagtg gtgcatggtg gtagtcccag 3360 atatttggaa ggctgaggcg ggaggatcgc ttgagcccag gaatttgagg ctgcagtgag 3420 ctgtgatcac accactgcac tccagcctca gtgacagagt gaggccctgt ctcaaaaaag 3480 aaaagaaaaa agaaaaataa tgagggctgt atggaataca ttcattattc attcactcac 3540 tcactcactc attcattcat tcattcattc aacaagtctt attgcatacc ttctgtttgc 3600 tcagcttggt gcttggggct gctgaggggc aggagggaga gggtgacatg ggtcagctga 3660 ctcccagagt ccactccctg taggtcgggc agcaggccgt agaagtctgg cagggcctgg 3720 ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt ggtcaactct tcccagccgt 3780 gggagcccct gcagctgcat gtggataaag ccgtcagtgg ccttcgcagc ctcaccactc 3840 tgcttcgggc tctgggagcc caggtgagta ggagcggaca cttctgcttg ccctttctgt 3900 aagaagggga gaagggtctt gctaaggagt acaggaactg tccgtattcc ttccctttct 3960 gtggcactgc agcgacctcc tgttttctcc ttggcagaag gaagccatct cccctccaga 4020 tgcggcctca gctgctccac tccgaacaat cactgctgac actttccgca aactcttccg 4080 agtctactcc aatttcctcc ggggaaagct gaagctgtac acaggggagg cctgcaggac 4140 aggggacaga tgaccagctt gatatcgaat tatccatcac actggcggcc gctcgagtct 4200 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 4260 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 4320 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 4380 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 4440 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 4500 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4560 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 4620 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 4680 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 4740 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 4800 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 4860 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 4920 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 4980 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 5040 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 5100 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 5160 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 5220 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 5280 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 5340 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 5400 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 5460 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 5520 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 5580 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 5640 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 5700 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 5760 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 5820 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 5880 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 5940 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 6000 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 6060 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 6120 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 6180 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 6240 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 6300 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 6360 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 6420 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 6480 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 6540 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 6600 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 6660 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 6720 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 6780 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 6840 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 6900 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 6960 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 7020 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 7080 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 7140 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 7200 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 7260 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 7320 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 7380 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca 7440 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 7500 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 7560 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 7620 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 7680 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 7740 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 7800 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 7860 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 7920 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 7980 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 8040 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 8100 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 8160 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 8220 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 8280 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 8340 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 8400 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 8460 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 8520 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 8580 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc 8635 <210> 3 <211> 8657 <212> DNA <213> Artificial Sequence <220> <223> X3GC / GEPO vector <400> 3 gacggatcgg gagatcccta actccgccca gttccgccca ttctccgccc catggctgac 60 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt 120 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga tggttcgacc 180 attgaactgc atcgtcgccg tgtcccaaaa tatggggatt ggcaagaacg gaaacctacc 240 ctggcctccg ctcaggaacg agttcaagta cttccaaaga atgaccacaa cctcttcagt 300 ggaaggtaaa cagaatctgg tgattatggg taggaaaacc tggttctcca ttcctgagaa 360 gaatcgacct ttaaaggaca gaattaatat agttctcagt agagaactca aagaaccacc 420 acgaggagct cattttcttg ccaaaagttt ggatgatgcc ttaagactta ttgaacaacc 480 ggaattggca agtaaagtag acatggtttg gatagtcgga ggcagttctg tttaccagga 540 agccatgaat caaccaggcc acctcagact ctttgtgaca aggatcatgc aggaatttga 600 aagtgacacg tttttcccag aaattgattt ggggaaatat aaacttctcc cagaataccc 660 aggcgtcctc tctgaggtcc aggaggaaaa aggcatcaag tataagtttg aagtctacga 720 gaagaaagac taacaggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 780 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 840 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact 900 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 960 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 1020 atgtctggga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc 1080 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag 1140 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag 1200 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt 1260 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 1320 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 1380 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 1440 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 1500 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 1560 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 1620 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 1680 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 1740 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 1800 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc 1860 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt 1920 aaacttaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt 1980 ctgcagattc gaggtcgacg gtatcgataa gcttccaggc gcggagatgg gggtgcacgg 2040 tgagtactcg cgggctgggc gctcccgccc gcccgggtcc ctgtttgagc ggggatttag 2100 cgccccggct attggccagg aggtggctgg gttcaaggac cggcgacttg tcaaggaccc 2160 cggaaggggg aggggggtgg ggcagcctcc acgtgccagc ggggacttgg gggagtcctt 2220 ggggatggca aaaacctgac ctgtgaaggg gacacagttt gggggttgag gggaagaagg 2280 tttgggggtt ctgctgtgcc agtggagagg aagctgataa gctgataacc tgggcgctgg 2340 agccaccact tatctgccag aggggaagcc tctgtcacac caggattgaa gtttggccgg 2400 agaagtggat gctggtagct gggggtgggg tgtgcacacg gcagcaggat tgaatgaagg 2460 ccagggaggc agcacctgag tgcttgcatg gttggggaca ggaaggacga gctggggcag 2520 agacgtgggg atgaaggaag ctgtccttcc acagccaccc ttctccctcc ccgcctgact 2580 ctcagcctgg ctatctgttc tagaatgtcc tgcctggctg tggcttctcc tgtccctgct 2640 gtcgctccct ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacagccg 2700 agtcctggag aggtacctct tggaggccaa ggaggccgag aatatcacgg tgagacccct 2760 tccccagcac attccacaga actcacgctc agggcttcag ggaactcctc ccagatccag 2820 gaacctggca cttggtttag ggtggagttg ggaagctaga cactgccccc ctacataaga 2880 ataagtctgg tggccccaaa ccatacctgg aaactaggca aggagcaaag ccagcagatc 2940 ctacggcctg tgggccaggg ccagagcctt cagggaccct tgactccccg ggctgtgtgc 3000 atttcagacg ggctgtgctg aacactgcag cttgaatgag aatatcactg tcccagacac 3060 caaagttaat ttctatgcct ggaagaggat ggaggtgagt tccttttttt ttttttttcc 3120 tttcttttgg agaatctcat ttgcgagcct gattttggat gaaagggaga atgatcgagg 3180 gaaaggtaaa atggagcagc agagatgagg ctgcctgggc gcagaggctc acgtctataa 3240 tcccaggctg agatggccga gatgggagaa ttgcttgagc cctggagttt cagaccaacc 3300 taggcagcat agtgagatcc cccatctcta caaacattta aaaaaattag tcaggtgaag 3360 tggtgcatgg tggtagtccc agatatttgg aaggctgagg cgggaggatc gcttgagccc 3420 aggaatttga ggctgcagtg agctgtgatc acaccactgc actccagcct cagtgacaga 3480 gtgaggccct gtctcaaaaa agaaaagaaa aaagaaaaat aatgagggct gtatggaata 3540 cattcattat tcattcactc actcactcac tcattcattc attcattcat tcaacaagtc 3600 ttattgcata ccttctgttt gctcagcttg gtgcttgggg ctgctgaggg gcaggaggga 3660 gagggtgaca tgggtcagct gactcccaga gtccactccc tgtaggtcgg gcagcaggcc 3720 gtagaagtct ggcagggcct ggccctgctg tcggaagctg tcctgcgggg ccaggccctg 3780 ttggtcaact cttcccagcc gtgggagccc ctgcagctgc atgtggataa agccgtcagt 3840 ggccttcgca gcctcaccac tctgcttcgg gctctgggag cccaggtgag taggagcgga 3900 cacttctgct tgccctttct gtaagaaggg gagaagggtc ttgctaagga gtacaggaac 3960 tgtccgtatt ccttcccttt ctgtggcact gcagcgacct cctgttttct ccttggcaga 4020 aggaagccat ctcccctcca gatgcggcct cagctgctcc actccgaaca atcactgctg 4080 acactttccg caaactcttc cgagtctact ccaatttcct ccggggaaag ctgaagctgt 4140 acacagggga ggcctgcagg acaggggaca gatgaccagc ttgatatcga attatccatc 4200 acactggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt 4260 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 4320 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 4380 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 4440 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 4500 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 4560 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4620 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4680 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4740 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4800 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 4860 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 4920 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta 4980 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 5040 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 5100 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta 5160 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 5220 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga 5280 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa 5340 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 5400 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 5460 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 5520 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 5580 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 5640 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 5700 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 5760 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 5820 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 5880 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 5940 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 6000 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 6060 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 6120 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 6180 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 6240 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 6300 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 6360 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 6420 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 6480 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 6540 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 6600 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 6660 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 6720 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 6780 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 6840 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 6900 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 6960 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 7020 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 7080 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 7140 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 7200 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 7260 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 7320 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 7380 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 7440 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 7500 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 7560 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 7620 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 7680 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 7740 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 7800 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 7860 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 7920 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 7980 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 8040 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 8100 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 8160 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 8220 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 8280 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 8340 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 8400 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 8460 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 8520 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 8580 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 8640 aagtgccacc tgacgtc 8657 <210> 4 <211> 8691 <212> DNA <213> Artificial Sequence <220> <223> x6GC / GEPO vector <400> 4 gacggatcgg gagatcccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 60 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 120 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 180 ggcttttgca aaaagctccc gggatggttc gaccattgaa ctgcatcgtc gccgtgtccc 240 aaaatatggg gattggcaag aacggaaacc taccctggcc tccgctcagg aacgagttca 300 agtacttcca aagaatgacc acaacctctt cagtggaagg taaacagaat ctggtgatta 360 tgggtaggaa aacctggttc tccattcctg agaagaatcg acctttaaag gacagaatta 420 atatagttct cagtagagaa ctcaaagaac caccacgagg agctcatttt cttgccaaaa 480 gtttggatga tgccttaaga cttattgaac aaccggaatt ggcaagtaaa gtagacatgg 540 tttggatagt cggaggcagt tctgtttacc aggaagccat gaatcaacca ggccacctca 600 gactctttgt gacaaggatc atgcaggaat ttgaaagtga cacgtttttc ccagaaattg 660 atttggggaa atataaactt ctcccagaat acccaggcgt cctctctgag gtccaggagg 720 aaaaaggcat caagtataag tttgaagtct acgagaagaa agactaacag gttcgaaatg 780 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 840 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 900 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 960 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 1020 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gggatctccc gatcccctat 1080 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg 1140 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag 1200 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg 1260 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 1320 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 1380 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 1440 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 1500 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 1560 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 1620 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 1680 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 1740 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 1800 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 1860 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga 1920 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttggta ccgagctcgg 1980 atccactagt aacggccgcc agtgtgctgg aattctgcag attcgaggtc gacggtatcg 2040 ataagcttcc aagcgcggag atgggggtgc acggtgagta ctcgcgggct gggcgctccc 2100 gcccgcccgg gtccctgttt gagcggggat ttagcgcccc ggctattggc caggaggtgg 2160 ctgggttcaa ggaccggcga cttgtcaagg accccggaag ggggaggggg gtggggcagc 2220 ctccacgtgc cagcggggac ttgggggagt ccttggggat ggcaaaaacc tgacctgtga 2280 aggggacaca gtttgggggt tgaggggaag aaggtttggg ggttctgctg tgccagtgga 2340 gaggaagctg ataagctgat aacctgggcg ctggagccac cacttatctg ccagagggga 2400 agcctctgtc acaccaggat tgaagtttgg ccggagaagt ggatgctggt agctgggggt 2460 ggggtgtgca cacggcagca ggattgaatg aaggccaggg aggcagcacc tgagtgcttg 2520 catggttggg gacaggaagg acgagctggg gcagagacgt ggggatgaag gaagctgtcc 2580 ttccacagcc acccttctcc ctccccgcct gactctcagc ctggctatct gttctagaat 2640 gtcctgcctg gctgtggctt ctcctgtccc tgctgtcgct ccctctgggc ctcccagtcc 2700 tgggcgcccc accacgcctc atctgtgaca gccgagtcct ggagaggtac ctcttggagg 2760 ccaaggaggc cgagaatatc acggtgagac cccttcccca gcacattcca cagaactcac 2820 gctcagggct tcagggaact cctcccagat ccaggaacct ggcacttggt ttagggtgga 2880 gttgggaagc tagacactgc ccccctacat aagaataagt ctggtggccc caaaccatac 2940 ctggaaacta ggcaaggagc aaagccagca gatcctacgg cctgtgggcc agggccagag 3000 ccttcaggga cccttgactc cccgggctgt gtgcatttca gacgggctgt gctgaacact 3060 gcagcttgaa tgagaatatc actgtcccag acaccaaagt taatttctat gcctggaaga 3120 ggatggaggt gagttccttt tttttttttt ttcctttctt ttggagaatc tcatttgcga 3180 gcctgatttt ggatgaaagg gagaatgatc gagggaaagg taaaatggag cagcagagat 3240 gaggctgcct gggcgcagag gctcacgtct ataatcccag gctgagatgg ccgagatggg 3300 agaattgctt gagccctgga gtttcagacc aacctaggca gcatagtgag atcccccatc 3360 tctacaaaca tttaaaaaaa ttagtcaggt gaagtggtgc atggtggtag tcccagatat 3420 ttggaaggct gaggcgggag gatcgcttga gcccaggaat ttgaggctgc agtgagctgt 3480 gatcacacca ctgcactcca gcctcagtga cagagtgagg ccctgtctca aaaaagaaaa 3540 gaaaaaagaa aaataatgag ggctgtatgg aatacattca ttattcattc actcactcac 3600 tcactcattc attcattcat tcattcaaca agtcttattg cataccttct gtttgctcag 3660 cttggtgctt ggggctgctg aggggcagga gggagagggt gacatgggtc agctgactcc 3720 cagagtccac tccctgtagg tcgggcagca ggccgtagaa gtctggcagg gcctggccct 3780 gctgtcggaa gctgtcctgc ggggccaggc cctgttggtc aactcttccc agccgtggga 3840 gcccctgcag ctgcatgtgg ataaagccgt cagtggcctt cgcagcctca ccactctgct 3900 tcgggctctg ggagcccagg tgagtaggag cggacacttc tgcttgccct ttctgtaaga 3960 aggggagaag ggtcttgcta aggagtacag gaactgtccg tattccttcc ctttctgtgg 4020 cactgcagcg acctcctgtt ttctccttgg cagaaggaag ccatctcccc tccagatgcg 4080 gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact cttccgagtc 4140 tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg caggacaggg 4200 gacagatgac cagcttgata tcgaattatc catcacactg gcggccgctc gagtctagag 4260 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 4320 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 4380 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 4440 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 4500 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 4560 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 4620 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 4680 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 4740 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 4800 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 4860 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 4920 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 4980 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 5040 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 5100 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 5160 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 5220 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga 5280 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc 5340 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat 5400 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 5460 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 5520 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 5580 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 5640 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 5700 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 5760 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 5820 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 5880 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 5940 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 6000 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 6060 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 6120 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 6180 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 6240 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 6300 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 6360 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 6420 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 6480 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 6540 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 6600 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 6660 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 6720 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 6780 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 6840 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 6900 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 6960 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 7020 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 7080 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 7140 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 7200 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 7260 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 7320 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 7380 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 7440 tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt 7500 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 7560 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 7620 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 7680 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 7740 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 7800 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 7860 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 7920 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 7980 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 8040 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 8100 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 8160 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 8220 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 8280 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 8340 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 8400 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 8460 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 8520 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 8580 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 8640 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 8691 <210> 5 <211> 7022 <212> DNA <213> Artificial Sequence <220> <223> X0GC / dhfr vector <400> 5 gacggatcgg gagatccgac atgataagat acattgatga gtttggacaa accacaacta 60 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 120 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 180 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg 240 ctgattatga tctctagtca aggcactata catcaaatat tccttattaa cccctttaca 300 aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag acactctatg 360 cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc tacttataaa 420 ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt cctttgtggt 480 gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca aaaaacttag 540 caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg gaggagtaga 600 atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg agcaaaacag 660 gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat aggttggaat 720 ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta aaaattttat 780 atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca caccacagaa 840 gtaaggttcc ttcacaaaga tccaaagcca gcaaaagtcc catggtctta taaaaatgca 900 tagctttagg aggggagcag agaacttgaa agcatcttcc tgttagtctt tcttctcgta 960 gacttcaaac ttatacttga tgcctttttc ctcctggacc tcagagagga cgcctgggta 1020 ttctgggaga agtttatatt tccccaaatc aatttctggg aaaaacgtgt cactttcaaa 1080 ttcctgcatg atccttgtca caaagagtct gaggtggcct ggttgattca tggcttcctg 1140 gtaaacagaa ctgcctccga ctatccaaac catgtctact ttacttgcca attccggttg 1200 ttcaataagt cttaaggcat catccaaact tttggcaaga aaatgagctc ctcgtggtgg 1260 ttctttgagt tctctactga gaactatatt aattctgtcc tttaaaggtc gattcttctc 1320 aggaatggag aaccaggttt tcctacccat aatcaccaga ttctgtttac cttccactga 1380 agaggttgtg gtcattcttt ggaagtactt gaactcgttc ctgagcggag gccagggtag 1440 gtctccgttc ttgccaatcc ccatattttg ggacacggcg acgatgcagt tcaatggtcg 1500 aaccatgatg gcagcgggga taaaatccta ccagccttca cgctaggatt gccgtcaagt 1560 ttggcgcgaa atcgcagccc tgagctgtgg atctcccgat cccctatggt gcactctcag 1620 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 1680 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 1740 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 1800 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 1860 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 1920 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 1980 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 2040 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 2100 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 2160 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 2220 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 2280 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 2340 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg 2400 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga 2460 gacccaagct tggtaccgag ctcggatcca ctagtaacgg ccgccagtgt gctggaattc 2520 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata 2580 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 2640 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 2700 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 2760 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 2820 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 2880 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 2940 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 3000 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 3060 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 3120 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 3180 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 3240 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 3300 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 3360 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 3420 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 3480 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 3540 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 3600 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 3660 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg 3720 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 3780 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 3840 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 3900 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 3960 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 4020 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 4080 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 4140 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 4200 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 4260 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 4320 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 4380 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 4440 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 4500 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 4560 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 4620 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 4680 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 4740 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 4800 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 4860 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 4920 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 4980 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5040 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5100 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5160 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5220 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5280 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5340 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5400 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5460 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5520 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5580 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5640 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5700 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5760 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5820 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5880 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5940 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 6000 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 6060 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6120 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6180 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 6240 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6300 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6360 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6420 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6480 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6540 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6600 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6660 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6720 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6780 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6840 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6900 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6960 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 7020 tc 7022 <210> 6 <211> 6422 <212> DNA <213> Artificial Sequence <220> <223> X1GC / dhfr vector <400> 6 gacggatcgg gagatccatt ctccgcccca tggctgacta atttttttta tttatgcaga 60 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 120 cctaggcttt tgcaaaaagc tcccgggatg gttcgaccat tgaactgcat cgtcgccgtg 180 tcccaaaata tggggattgg caagaacgga aacctaccct ggcctccgct caggaacgag 240 ttcaagtact tccaaagaat gaccacaacc tcttcagtgg aaggtaaaca gaatctggtg 300 attatgggta ggaaaacctg gttctccatt cctgagaaga atcgaccttt aaaggacaga 360 attaatatag ttctcagtag agaactcaaa gaaccaccac gaggagctca ttttcttgcc 420 aaaagtttgg atgatgcctt aagacttatt gaacaaccgg aattggcaag taaagtagac 480 atggtttgga tagtcggagg cagttctgtt taccaggaag ccatgaatca accaggccac 540 ctcagactct ttgtgacaag gatcatgcag gaatttgaaa gtgacacgtt tttcccagaa 600 attgatttgg ggaaatataa acttctccca gaatacccag gcgtcctctc tgaggtccag 660 gaggaaaaag gcatcaagta taagtttgaa gtctacgaga agaaagacta acaggttcga 720 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 780 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 840 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg 900 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 960 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgggatc tcccgatccc 1020 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc 1080 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg 1140 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt 1200 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa 1260 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 1320 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 1380 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 1440 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 1500 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 1560 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 1620 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 1680 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 1740 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 1800 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat 1860 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt ggtaccgagc 1920 tcggatccac tagtccagtg tggtggaatt ctgcagatat ccagcacagt ggcggccgct 1980 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 2040 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2100 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2160 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 2220 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 2280 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2340 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 2400 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 2460 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2520 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2580 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 2640 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 2700 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 2760 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 2820 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 2880 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 2940 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 3000 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 3060 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga 3120 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 3180 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 3240 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 3300 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 3360 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 3420 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 3480 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 3540 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 3600 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 3660 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 3720 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 3780 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 3840 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 3900 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga 3960 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 4020 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 4080 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 4140 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 4200 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt 4260 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 4320 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 4380 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 4440 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 4500 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 4560 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 4620 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4680 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4740 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4800 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4860 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4920 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4980 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 5040 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 5100 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 5160 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca 5220 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5280 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5340 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5400 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5460 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5520 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5580 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5640 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5700 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 5760 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 5820 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 5880 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 5940 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6000 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6060 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6120 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6180 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6240 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6300 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6360 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 6420 tc 6422 <210> 7 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> x0GC / dhfr cassette <400> 7 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 60 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 120 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 180 gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta tgatctctag 240 tcaaggcact atacatcaaa tattccttat taaccccttt acaaattaaa aagctaaagg 300 tacacaattt ttgagcatag ttattaatag cagacactct atgcctgtgt ggagtaagaa 360 aaaacagtat gttatgatta taactgttat gcctacttat aaaggttaca gaatattttt 420 ccataatttt cttgtatagc agtgcagctt tttcctttgt ggtgtaaata gcaaagcaag 480 caagagttct attactaaac acagcatgac tcaaaaaact tagcaattct gaaggaaagt 540 ccttggggtc ttctaccttt ctcttctttt ttggaggagt agaatgttga gagtcagcag 600 tagcctcatc atcactagat ggcatttctt ctgagcaaaa caggttttcc tcattaaagg 660 cattccacca ctgctcccat tcatcagttc cataggttgg aatctaaaat acacaaacaa 720 ttagaatcag tagtttaaca cattatacac ttaaaaattt tatatttacc ttagagcttt 780 aaatctctgt aggtagtttg tccaattatg tcacaccaca gaagtaaggt tccttcacaa 840 agatccaaag ccagcaaaag tcccatggtc ttataaaaat gcatagcttt aggaggggag 900 cagagaactt gaaagcatct tcctgttagt ctttcttctc gtagacttca aacttatact 960 tgatgccttt ttcctcctgg acctcagaga ggacgcctgg gtattctggg agaagtttat 1020 atttccccaa atcaatttct gggaaaaacg tgtcactttc aaattcctgc atgatccttg 1080 tcacaaagag tctgaggtgg cctggttgat tcatggcttc ctggtaaaca gaactgcctc 1140 cgactatcca aaccatgtct actttacttg ccaattccgg ttgttcaata agtcttaagg 1200 catcatccaa acttttggca agaaaatgag ctcctcgtgg tggttctttg agttctctac 1260 tgagaactat attaattctg tcctttaaag gtcgattctt ctcaggaatg gagaaccagg 1320 ttttcctacc cataatcacc agattctgtt taccttccac tgaagaggtt gtggtcattc 1380 tttggaagta cttgaactcg ttcctgagcg gaggccaggg taggtctccg ttcttgccaa 1440 tccccatatt ttgggacacg gcgacgatgc agttcaatgg tcgaaccatg atggcagcgg 1500 ggataaaatc ctaccagcct tcacgctagg attgccgtca agtttggcgc gaaatcgcag 1560 ccctgagctg tg 1572 <210> 8 <211> 988 <212> DNA <213> Artificial Sequence <220> <223> x1GC dhfr cassette <400> 8 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 60 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 120 agctcccggg atggttcgac cattgaactg catcgtcgcc gtgtcccaaa atatggggat 180 tggcaagaac ggaaacctac cctggcctcc gctcaggaac gagttcaagt acttccaaag 240 aatgaccaca acctcttcag tggaaggtaa acagaatctg gtgattatgg gtaggaaaac 300 ctggttctcc attcctgaga agaatcgacc tttaaaggac agaattaata tagttctcag 360 tagagaactc aaagaaccac cacgaggagc tcattttctt gccaaaagtt tggatgatgc 420 cttaagactt attgaacaac cggaattggc aagtaaagta gacatggttt ggatagtcgg 480 aggcagttct gtttaccagg aagccatgaa tcaaccaggc cacctcagac tctttgtgac 540 aaggatcatg caggaatttg aaagtgacac gtttttccca gaaattgatt tggggaaata 600 taaacttctc ccagaatacc caggcgtcct ctctgaggtc caggaggaaa aaggcatcaa 660 gtataagttt gaagtctacg agaagaaaga ctaacaggtt cgaaatgacc gaccaagcga 720 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 780 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 840 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 900 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 960 aactcatcaa tgtatcttat catgtctg 988 <210> 9 <211> 1010 <212> DNA <213> Artificial Sequence <220> X3GC dhfr cassette <400> 9 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 60 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 120 ggaggcctag gcttttgcaa aaagctcccg ggatggttcg accattgaac tgcatcgtcg 180 ccgtgtccca aaatatgggg attggcaaga acggaaacct accctggcct ccgctcagga 240 acgagttcaa gtacttccaa agaatgacca caacctcttc agtggaaggt aaacagaatc 300 tggtgattat gggtaggaaa acctggttct ccattcctga gaagaatcga cctttaaagg 360 acagaattaa tatagttctc agtagagaac tcaaagaacc accacgagga gctcattttc 420 ttgccaaaag tttggatgat gccttaagac ttattgaaca accggaattg gcaagtaaag 480 tagacatggt ttggatagtc ggaggcagtt ctgtttacca ggaagccatg aatcaaccag 540 gccacctcag actctttgtg acaaggatca tgcaggaatt tgaaagtgac acgtttttcc 600 cagaaattga tttggggaaa tataaacttc tcccagaata cccaggcgtc ctctctgagg 660 tccaggagga aaaaggcatc aagtataagt ttgaagtcta cgagaagaaa gactaacagg 720 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 780 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 840 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 900 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 960 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 1010 <210> 10 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> x6GC dhfr cassette <400> 10 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 60 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc 120 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct 180 cccgggatgg ttcgaccatt gaactgcatc gtcgccgtgt cccaaaatat ggggattggc 240 aagaacggaa acctaccctg gcctccgctc aggaacgagt tcaagtactt ccaaagaatg 300 accacaacct cttcagtgga aggtaaacag aatctggtga ttatgggtag gaaaacctgg 360 ttctccattc ctgagaagaa tcgaccttta aaggacagaa ttaatatagt tctcagtaga 420 gaactcaaag aaccaccacg aggagctcat tttcttgcca aaagtttgga tgatgcctta 480 agacttattg aacaaccgga attggcaagt aaagtagaca tggtttggat agtcggaggc 540 agttctgttt accaggaagc catgaatcaa ccaggccacc tcagactctt tgtgacaagg 600 atcatgcagg aatttgaaag tgacacgttt ttcccagaaa ttgatttggg gaaatataaa 660 cttctcccag aatacccagg cgtcctctct gaggtccagg aggaaaaagg catcaagtat 720 aagtttgaag tctacgagaa gaaagactaa caggttcgaa atgaccgacc aagcgacgcc 780 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 840 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 900 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 960 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 1020 catcaatgta tcttatcatg tctg 1044 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> 223 dhfr 01 primer <400> 11 gcgcccggga tggttcgacc attgaactgc 30 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> dhfr-02 primer <400> 12 cacttagaac ctgttagtct ttcttctcgt agac 34 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> x 1GC primer <400> 13 tcaggatcca ttctccgccc catggctgac taa 33 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> x 3GC primer <400> 14 catggatcct aactccgccc agttccgccc attct 35 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> x6GC primer <400> 15 catggatccc atagtcccgc ccctaactcc gccc 34 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> X0GC primer <400> 16 cgatggatcc gacatgataa gatacattga t 31 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> X0GCRR primer <400> 17 cgttggatcc acagctcagg gctgcgattt c 31 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> BISVpAR primer <400> 18 tcaggatccc agacatgata agatacattg atg 33  

Claims (20)

A high expression induction cassette having a nucleotide sequence from which at least one CCGCCC repeat sequence is removed from a GC-rich region between bases 8 to 67 of the nucleotide sequence defined by SEQ ID NO. The high expression induction cassette of claim 1, wherein the GC-rich region comprises up to one CCGCCC repeat sequence. A high expression induction cassette having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 10. An expression vector comprising the high expression induction cassette of any one of claims 1 to 3. The expression vector of claim 4, wherein the gene encoding the recombinant protein of interest is further operably linked to a high expression induction cassette. The expression vector according to claim 5, wherein the recombinant protein of interest is human erythropoietin. The expression vector according to claim 6, which has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4. The expression vector according to claim 4, which has a nucleotide sequence of SEQ ID NO: 5 or 6. A cell line transformed with the expression vector of claim 8. The cell line according to claim 9, which is accession number KCTC10991BP or KCTC10992BP. A cell line transformed with the expression vector of claim 5. 12. The cell line of claim 11, wherein said cell line is a CHO cell line. 13. The cell line according to claim 12, wherein the CHO cell line is deficient in the dihydrofolate reductase gene. The cell line according to claim 12, which is accession number KCTC10993BP, KCTC10994BP or KCTC10995BP. (a) transforming an animal cell line with an expression vector comprising the high expression induction cassette of any one of claims 1 to 3; And (b) culturing the transformed animal cell line in the presence of a dihydrofolate reductase inhibitor. The method of claim 15, wherein the recombinant protein of interest is human erythropoietin. The method of claim 16, further comprising purifying the sialic acid-containing erythropoietin. The method of claim 15, wherein the expression vector is the expression vector described in FIG. 1. The method of claim 15, wherein the animal cell line of step (a) is a dihydrofolate reductase deficient CHO cell line. The method of claim 15, wherein the transformed animal cell line of step (b) is an animal cell line with accession number KCTC10993BP, KCTC10994BP or KCTC10995BP.

Images